monomac 1 cells  (DSMZ)

Journal: Science (New York, N.Y.)

Article Title: Lipid presentation by the protein C receptor links coagulation with autoimmunity

doi: 10.1126/science.abc0956

Figure Lengend Snippet: (A) EPCR-dependent induction of IFN-regulated genes and Tnf in mouse monocytes after 1 hour of stimulation with LPS, aPL HL5B, aPL HL7G, or TLR7 agonist R848; relative expression induced by aPLs was normalized to IgG isotype control; n=6, *P<0.05 compared to stimulation without inhibitor; two-way ANOVA, Sidak’s multiple comparisons test. (B) Volcano plot of aPL HL7G-induced transcripts, including Stat1, a known IFN-regulated gene. (C) Induction of Tnf in CD115+ splenic monocytes from ProcrC/S or strain matched ProcrWT mice (upper panel) and human trophoblast cells (lower panel) stimulated for 1 or 3 hours with IgG (100 µg/ml) isolated from APS patients with confirmed reactivity to cardiolipin alone (α-CL), β2GPI alone (α-β2GPI), or dual reactivity (α-CL/β2GP). Human trophoblast cells were pretreated with either non-inhibitory α-EPCR 1489 or inhibitory α-EPCR 1496; *P<0.0001; one-way ANOVA. (D) Live-cell imaging of aPL HL5B IgG or F(ab′)2 colocalization (green) with cholera toxin B (CTB; magenta) or EPCR (blue) using non-inhibitory α-EPCR 1489 in human MM1 cells. Nuclei were stained with Hoechst 33342 (gray). Quantification of colocalization; n=3 ROI (regions of interest) consisting of at least three cells, *P<0.0001; one-way ANOVA. (E) Live-cell imaging of HL5B internalization (green) in monocytes of the indicated mouse strains with CTB or LysoTracker counterstaining (magenta). Nuclei were stained with Hoechst 33342 (blue); bar=5 µm; n=3 ROI, *P<0.0001; one-way ANOVA.

Article Snippet: aPL signaling was evaluated in established monocyte, MonoMac-1 (MM1) (DSMZ, ACC252), endothelial, and trophoblast (JAR) cell (DSMZ, ACC462) models ( 11 , 12 , 15 ) cultured in RPMI, 10% FCS.

Techniques: Expressing, Control, Isolation, Live Cell Imaging, Staining

Journal: Science (New York, N.Y.)

Article Title: Lipid presentation by the protein C receptor links coagulation with autoimmunity

doi: 10.1126/science.abc0956

Figure Lengend Snippet: (A) aPL-mediated phosphatidylserine exposure (measured by Annexin V-FITC staining) and aPL-mediated TF activation in MM1 cells (measured as procoagulant activity, PCA) were prevented by desipramine; n=6, *P<0.0001; one-way ANOVA. (B) aPL internalization in MM1 cells is blocked by sphingomyelinase inhibitor desipramine. Bar=5 µm. Quantification of colocalization; n=3 ROI consisting of at least three cells, *P=0.002; t-test. (C) aPL-induced ASM activity in MM1 cells is blocked by inhibitors of FXa (Rivaroxaban, NAP5), thrombin (hirudin), and PAR1 cleavage (αPAR1, ATAP2/WEDE15) but not by inhibitors of complement (compstatin), PDI and ARF6; n=3, *P<0.0001; one-way ANOVA. (D) Surface ASM exposure in MM1 cells after 30 min of stimulation with aPL HL5B F(ab′)2. Scale bar=5 µm. Live-cell imaging of EPCR colocalization (green) with cholera toxin B (CTB; magenta) or ASM (blue). Nuclei were stained with Hoechst 33342 (gray). (E) LBPA (10 µM) loading of mouse ProcrC/S cells enables ASM activation in CD115+ monocytes stimulated with HL5B; n=3, *P<0.0001; one-way ANOVA. (F) ASM activity in unstimulated mouse monocytes lysates after addition of sEPCR–LBPA (2.5 µM) is blocked by α-EPCR–LBPA 1682; n=3, *P<0.0001; one-way ANOVA. (G, H) Proximity ligation assays (PLA) with magenta fluorescence dots for ASM and EPCR on ALIX−/− trophoblast cells after 10 min of stimulation with aPL HL5B F(ab′)2 fragments (G) or thrombin (H) with or without LBPA loading. Nuclei were stained with Hoechst 33342 (blue) Scale bar=25 µm. (I) aPL HL5B signaling in ProcrC/S monocytes was restored by adding 10 µM LBPA (S,R), but not by LBPA (S,S) or hemi LBPA. Stimulation time 3 hours; n=6, *P<0.0001; one-way ANOVA. (J) aPL HL5B internalization in ALIX-deficient JAR cells after pretreatment with the indicated phospholipids at 10 µM. Scale bar=5 µm.

Article Snippet: aPL signaling was evaluated in established monocyte, MonoMac-1 (MM1) (DSMZ, ACC252), endothelial, and trophoblast (JAR) cell (DSMZ, ACC462) models ( 11 , 12 , 15 ) cultured in RPMI, 10% FCS.

Techniques: Staining, Activation Assay, Activity Assay, Live Cell Imaging, Ligation, Fluorescence

Journal: Science (New York, N.Y.)

Article Title: Lipid presentation by the protein C receptor links coagulation with autoimmunity

doi: 10.1126/science.abc0956

Figure Lengend Snippet: (A) Mice of the indicated genotypes were immunized with aPL HL5B and anti-cardiolipin titer determined at the indicated times; n=10, *P<0.0001 for ProcrC/S vs. WT; one-way ANOVA. (B) IgG isolated 12 weeks after the start of aPL immunization were used to stimulate human MM1 cells for 1 hour for induction of the indicated genes; n=5, *P<0.05, **P=0.011, ***P<0.0001; two-way ANOVA, Sidak’s multiple comparisons test. (C) MRL-Faslpr lupus-prone mice were treated with the indicated α-EPCR antibodies at an age of 4 weeks (day 0) and anti-cardiolipin titers were determined in serum at the indicated time points; n=5, *P=0.03; **P<0.0001; two-way ANOVA, Sidak’s multiple comparisons test. (D) Antibodies to double stranded (ds) DNA were measured in α-EPCR 1650- and α-EPCR–LBPA 1682-treated MRL-Faslpr mice 2 weeks after the last dose or in 6-week-old MRL/MpJ control or MRL-Faslpr mice; n=4–5, *P<0.0001; one-way ANOVA. (E) Immune cell infiltration of α-EPCR-treated MRL-Faslpr mice; n=5, *P<0.025. (F) Renal pathology scores of α-EPCR-treated MRL-Faslpr mice; n=5, *P=0.0317; Mann–Whitney U test. Scale bars=80 µm.

Article Snippet: aPL signaling was evaluated in established monocyte, MonoMac-1 (MM1) (DSMZ, ACC252), endothelial, and trophoblast (JAR) cell (DSMZ, ACC462) models ( 11 , 12 , 15 ) cultured in RPMI, 10% FCS.

Techniques: Isolation, Control, MANN-WHITNEY